The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide-and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease. INTRODUCTIONThe increased prevalence of obesity worldwide has contributed to the emergence of a disease cluster, the metabolic syndrome, that includes insulin resistance, type 2 diabetes, and cardiovascular disease. Elevated serum triglyceride and fatty acid (FA) levels associated with this syndrome contribute to lipid accumulation in many nonadipose tissues, including the heart. This inappropriate accumulation of excess lipid can lead to cellular dysfunction and cell death-a process called lipotoxicity (Unger, 2003).Lipotoxic cardiomyocyte death has been proposed to play a central role in heart failure associated with diabetes and obesity in animal models and in humans. Although FAs are the principal source of energy for cardiomyocytes, high serum triglyceride and FA levels result in FA uptake by the heart that exceeds the anabolic and catabolic needs of the tissue. Triglyceride accumulation in cardiomyocytes of leptin-or leptin receptor-deficient obese diabetic animal models is associated with cardiomyocyte apoptosis (Zhou et al., 2000) and contractile dysfunction (Zhou et al., 2000;Aasum et al., 2002;Christoffersen et al., 2003), suggesting that lipotoxic cell death in the heart may be important in the genesis of diabetic cardiomyopathy. Recently, similar observations were reported in patients with metabolic syndrome and nonischemic heart failure (Sharma et al., 2004). Consistent with this apparent cardiac lipotoxicity, cardiomyocyte-specific increases in FA uptake in mice with cardiac-restricted overexpression of long-chain acyl-CoA synthetase 1 (ACS1), li...

To learn more about the targets of Cn (Cn) and calcium/calmodulin-dependent protein kinase in cardiac muscle, we investigated their actions in cultured cardiac myocytes and the hearts of mice in vivo. Adenoviral-mediated expression of constitutively active forms of either pathway induced expression of peroxisome proliferator-activated receptor ␥ coactivator 1␣, a transcriptional coactivator involved in the control of multiple cellular energy metabolic pathways in cardiac myocytes. Transcriptional profiling studies demonstrated that Cn and calcium/calmodulin-dependent protein kinase activate distinct but overlapping metabolic gene regulatory programs. Expression of the nuclear receptor, peroxisome proliferator-activated receptor ␣, was markedly increased by Cn, but not calcium/calmodulin-dependent protein kinase, providing one mechanism whereby cellular fatty acid utilization genes are selectively activated by Cn. Transfection experiments demonstrated that Cn directly activates the mouse peroxisome proliferator-activated receptor ␣ gene promoter. Co-transfection "add-back" experiments demonstrated that the transcription factors, myocyte enhancer factors 2C or 2D, were sufficient to confer Cn-mediated activation of the peroxisome proliferatoractivated receptor ␣ gene. Cn was also shown to directly activate a known peroxisome proliferator-activated receptor ␣ target, muscle-type carnitine palmitoyltransferase I, providing a second mechanism by which Cn activates genes of cellular fatty acid utilization. Lastly, the gene expression of peroxisome proliferator-activated receptor ␥ coactivator 1␣ and peroxisome proliferator-activated receptor ␣ was reduced in the hearts of mice with cardiac-specific ablation of the Cn regulatory subunit. These data support a role for calcium-triggered signaling pathways in the regulation of cardiac energetics and identify pathway-specific control of metabolic targets.Skeletal muscle fiber types are defined by contractile protein isoform composition and energy metabolic properties. Slowtwitch muscle fibers possess greater mitochondrial volume supporting higher oxidative metabolic capacity compared with fast-twitch fibers. Muscle metabolic phenotype is plastic, responding to numerous external stimuli. Calcium signaling serves an important role in the adaptive response of skeletal muscle to external stimuli, including fiber type determination. Evidence has emerged that calcium-triggered regulatory pathways acting through Cn (Cn), 1 a serine/threonine protein phosphatase, and calcium/calmodulin-dependent protein kinases (CaMK), serve a major role in determining the functional and metabolic phenotype of skeletal muscle by transducing alterations in cytosolic calcium concentration. In support of this, Chin et al. (1) demonstrated that a constitutively active form of Cn (Cn*) is capable of activating transcription of slow fiberspecific gene promoters. Similarly, overexpression of Cn* in skeletal muscle results in an increase in slow muscle fiber types (2), and in CnA␣ and A␤ null mice there ...